1. Field of the Invention
This invention relates generally to analytical tests for the determination of peroxidatively active substances in test samples, and particularly to an improved test composition and device for such determinations having enhanced storage stability, as well as to a method for making and using the improved composition and device.
Many analytical methods are available for detecting the presence of peroxidatively active substances in biological samples such as urine, fecal suspensions and gastrointestinal contents. Hemoglobin and its derivatives are typical examples of such "peroxidatively active" substances because they behave in a manner similar to the enzyme peroxidase. Such substances have also been referred to as pseudoperoxidases, i.e., enzyme-like in that they catalyze the redox reaction between peroxides and such indicator compounds as benzidine, o-tolidine, 3,3',5,5'-tetramethylbenzidine, 2,7-diaminofluorene or similar substances, thereby producing a detectable response such as a color change. Most methods for determining the presence of occult blood in test samples therefore rely on this pseudoperoxidase activity.
2. Background Art
Analytical test methods have evolved over the years which rely on enzyme-like catalysis of the peroxidative oxidation of colorforming indicators. These include wet chemical or solution procedures and the so-called "dip-and-read" type, reagent-bearing strip devices. Of the former, a typical example is set forth in Richard M. Henry, et al., Clinical Chemistry Principles and Techniques, 2nd ed., (Hagerstown, Md.: Harper and Row, 1974), pp. 1124-1125. This procedure involves the use of glacial acetic acid (buffer), diphenylamine (indicator) and hydrogen peroxide. While such wet methods have proven analytical utility, they nevertheless have shortcomings, such as poor reagent stability and inadequate sensitivity.
Another method for the determination of peroxidatively active substances, and one presently preferred by most clinical assayists, employs the "dip-and-read", solid phase reagent strip device. Typical of such devices are those commercially available from the Ames Division of Miles Laboratories, Inc. under the trademark HEMASTIX.RTM.. They comprise, in essence, a porous paper matrix affixed to a plastic strip or handle. The matrix is impregnated with a buffered mixture of an organic hydroperoxide, for example, cumene hydroperoxide, and an indicator compound. Upon immersion in a liquid containing an analyte such as hemoglobin, myoglobin, erythrocytes or other pseudoperoxidases, a blue color develops in the matrix, the intensity of which is proportional to the concentration of the peroxidatively active substance in the sample. Thus, by comparing the color developed in the matrix to a standard color chart, the assayist can determine, on a semiquantitative basis, the amount of analyte present in the sample.
Reagent strips possess a number of advantages over wet chemistry methods, for example: greater ease of use because neither the preparation of reagents nor attendant apparatus is required; greater comparative stability of reagents because of the dry, solid reagent state, resulting in improved accuracy, sensitivity and economy. However, a serious disadvantage of many conventional, presently-available reagent strip test devices has been limited "shelf-life", i.e., a lack of storage stability over prolonged periods following manufacture, resulting in markedly decreased reactivity to the presence of peroxidatively active analytes when the devices are used. Thus, because analytical tools such as reagent strips usually are not used immediately after manufacture, but stored for varying periods of time before use, and because too long a period between manufacture and use of conventional reagent strips can result in a severe loss of reactivity, leading to false negative test results, enhanced shelf-life can be a marked asset: the better the shelf-life, the more dependable the analytical results.
Conventional solid phase reagent strip devices for determining peroxidatively active substances, e.g., occult blood or hemoglobin in urine, typically utilize as an indicator system the porphyrin-catalyzed oxidation of a benzidine-type indicator, for example, o-tolidine or 3,3',5,5'-tetramethylbenzidine, by an organic hydroperoxide, such as cumene hydroperoxide. Such conventional test strips, however, are known to be particularly prone to loss of reactivity during prolonged storage, or storage at elevated temperatures--a phenomenom which is believed to be due either to volatility or chemical degradation of one or more reagent ingredients of the strip. Not only have substantial losses of reactivity been observed in such conventional reagent strips following storage at ambient temperatures, but those losses appear to be substantially accentuated, and the rate of loss accelerated, by storage at elevated temperatures. Possible explanations for the losses of reactivity in reagent strips are: (1) key ingredient(s) of the reagent composition decompose or volatilize, so that the level of ingredient(s) falls below the minimum level necessary to maintain adequate reactivity; and (2) two or more ingredients in the strip interact deleteriously, producing one or more new species which are unreactive or inhibitory.
Attempts to stabilize the reactivity of reagent compositions, and solid phase strip devices made therefrom for determining peroxidatively active substances, have followed various lines of approach. For example, U.S. Pat. No. 3,092,463 to Adams, Jr. et al., discloses an improved test composition and device for detecting occult blood in body fluids. The composition comprises an organic hydroperoxide encapsulated or entrapped in microspherical bubbles of a colloid substance, an indicator or dye precursor capable of accepting transfer of oxygen from the organic hydroperoxide to produce a color response induced by the catalytic action of the prosthetic group of hemoglobin, and a buffer for maintaining the pH of the substance being tested within the range of from 4 to 6.5. This patent discloses that the colloid substance, for example, polyvinyl alcohol, gelatin, gum arabic or carboxy vinyl polymer, used to encapsulate or entrap the hydroperoxide, can provide stabilization of the reactivity of preferred embodiments of the test device produced from the composition even after 300 hours storage at 75.degree. C., whereas similar devices prepared without encapsulation of the hydroperoxide lost reactivity after 24 hours at 50.degree. C.
Other disclosures of stabilized test compositions and devices include the approach of U.S. Pat. No. 3,252,762 to Adams, Jr. et al., wherein the organic hydroperoxide used is encapsulated in a colloidal material such as gelatin which is hardened by fixing with a dialdehyde polysaccharide. Such compositions, containing a hydroperoxide so encapsulated, a suitable indicator and a buffer, are said to exhibit enhanced stability under various adverse temperature conditions,
Still further disclosed attempts at stabilization of such reagent strip devices include a recitation in Chemical Abstracts, Vol. 85, p. 186 (1976), describing a two-dip method for preparing reagent strips containing o-tolidine and phenylisopropyl hydroperoxide. This disclosure reports making a solution of the indicator (o-tolidine.2HCl) and polyvinylpyrrolidone in ethanol. To this solution was added a small amount of surfactant and enough citrate buffer to provide a pH of 3.7, whereafter filter paper strips impregnated with ethyl cellulose were dipped in this solution and dried. The impregnated filter paper was subsequently dipped into a second solution containing 1,4-diazabicyclo [2,2,2]octane, phenylisopropyl hydroperoxide and polyvinyl pyrrolidone, dissolved in an ethanol-toluene mixture. The thrust of this work was to stabilize the peroxide and indicator combination through the use of the bicyclooctane derivative and the polyvinylpyrrolidone.
A similar method is disclosed in U.S. Pat. No. 3,853,471. This patent discloses the use of phosphoric or phosphonic acid amides where the substituent amido groups are primarily N-morpholine radicals.
Other approaches to stabilized reagent compositions include that of U.S. Pat. No. 4,071,317, wherein various diluent compounds, such as a mixture of dimethyl sulfone and N,N-dimethyl formamide, are employed along with cumene hydroperoxide and an indicator; of U.S. Pat. No. 4,071,318 (use of borate esters); and of U.S. Pat. No. 4,071,321 (use of both diluents and borate esters).
Another reference which is of interest to these general concepts is U.S. Pat. No. 3,236,850, directed toward stabilizing organic hydroperoxides used as catalysts and oxidizing agents. This reference discloses the use of primary, secondary or tertiary amine salts with organic peroxides, and is not directed to stability problems of solid phase reagent test strip devices.
Despite the inherent analytical advantages of solid phase reagent strip devices over wet chemistry procedures, and the foregoing exemplary advances in the art of stabilizing the reactivity of such strip devices, the stability characteristics of the latter, particularly in the case of devices for the determination of peroxidatively active substances, are in need of still further improvement. Whereas the properties of current solid phase, state-of-the-art compositions and devices for determining peroxidatively active substances are greatly enhanced over those of wet chemical methods, and over those of methods including no stability-enhancement techniques, it would nonetheless be greatly advantageous if even more stability during prolonged storage could be afforded, and more sensitivity to peroxidatively active analytes following such storage could be achieved, without the need for the addition of chemical substances, isolation of reagents by encapsulation, or similar relatively complicated and expensive treatments of such compositions and devices. For example, it would be a great advance in the art to provide suitable, direct substitutes for the well-known organic hydroperoxides which are conventionally used in solid phase test compositions and devices, substitutes which would render the reagent systems of these compositions and devices more stable during long-term storage.
Conventionally-used hydroperoxides include, for example, cumene hydroperoxide, t-butyl hydroperoxide, diisopropylbenzene hydroperoxide, 2,5-dimethylhexane-2,5-dihydroperoxide and paramenthane hydroperoxide. However, any suitable substitute for such compounds must not only be capable of participating similarly in the redox reaction with a peroxidatively active substance (analyte), in the presence of the conventional indicator systems used, to produce a detectable response such as a color change or change in the amount of light absorbed or reflected by the test composition, but also must exhibit such reactivity to an extent comparable to that of the conventional hydroperoxides typically used.
It has now been postulated that the frequently-observed losses of reactivity, leading to lack of storage stability or "shelf-life", of conventional solid phase reagent compositions and strip devices for determining peroxidatively active substances, may be attributable primarily to loss and/or chemical degradation of the organic hydroperoxide used in the reagent strip. Such loss or degradation could occur, for example, from decomposition or volatilization of the hydroperoxide or chemical interaction with other strip constituents. However, it is now believed that degradation due to decomposition or deleterious interaction may account for most reactivity losses. The mechanism causing such degradation is at the present unknown.